Solid materials with charged surfaces are used widely in the field of polynucleotide purification. These materials commonly include ion exchangers, which are usually employed in either of two application formats. In bind-elute mode, the polynucleotide and ion exchanger are equilibrated to conditions that allow the polynucleotide species of interest to bind. Contaminants that interact weakly or not at all with the charged surface fail to bind and are eliminated. Contaminants that interact more strongly than the polynucleotide bind more strongly. After washing to remove unbound contaminants, the column may be eluted by increasing the salt concentration. This permits fractionation of bound species in increasing order of the strength of their interaction with the ion exchanger, thereby achieving a high degree of polynucleotide purification. In flow-through mode, for example on a cation exchanger, the sample and ion exchanger are both equilibrated to conditions that prevent the polynucleotide from binding. Species that interact more strongly with the ion exchanger than the polynucleotide are bound and thereby removed, but species that bind more weakly than the polynucleotide flow through with it and persist as contaminants. Both modes are performed on charged surfaces presented in a variety of solid phase architectures, including porous or non-porous particles packed in columns, or added directly to large volume aqueous samples, or on monoliths or membranes. These different architectures confer different flow properties, capacity, and resolution, but the defining chemical features of flow-through or bind-elute chromatography are constant regardless of physical format. Both methods rely on the equilibration of the ion exchanger and sample to the same conditions before the sample is introduced to the column.
Some chromatography media embody combinations of charges with other types of chemical functionalities. These media are broadly known as mixed-modes or multimodal materials, and may variously combine charges with hydrophobic groups, metal affinity, groups, or chemical groups that favor formation of hydrogen bonds. They are operated under different chemical conditions depending on the nature of the secondary functionalities, but they are still operated in bind-elute and flow-through mode.
A small minority of multimodal materials have been described that combine charge with a physical functionality, for example, where a charged material also has the ability to sort species according to their size. One such example employs variable size exclusion functionality in combination with an electropositive surface [Hunter, A., J. Chromatogr. A 897:65-80 (2000); Hunter, A., J. Chromatogr. A 897:81-97 (2000); Hunter, A., J. Chromatogr. A 930:79-93 (2000); Hunter, A., J. Chromatogr. A 971:105-116 (2000)]. The method generally involves entry of proteins into particle pores in a size-dependent manner while exhibiting codependence on the charge on the protein, as well as the buffer conditions. Like other charged materials for fractionation, the method employs bind-elute and flow-through modes of operation.
Recently (Nian et al. J. Chromatogr. A 1282 (2013) 127-132.) a new mode of chromatography has been described called void exclusion chromatography. The material for practicing the method includes a column packed with electropositive particles (cf. Hunter supra), with the proviso that the sample volume applied to a column is not greater than the volume occupied by the space between the particles. The method was described for purification of antibodies.